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SUPPLEMENTAL METHODS, FIGURES and TABLES Cohesin Mutations Alter DNA Damage Repair and Chromatin Structure and Create Therapeuti

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SUPPLEMENTAL METHODS, FIGURES and TABLES Cohesin Mutations Alter DNA Damage Repair and Chromatin Structure and Create Therapeuti SUPPLEMENTAL METHODS, FIGURES and TABLES Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML Zuzana Tothova1,2*, Anne-Laure Valton3, Rebecca A. Gorelov2, Mounica Vallurupalli1,2, John M. Krill-Burger2, Amie Holmes4, Catherine C. LanDers2, J. Erika HayDu2, EDyta Malolepsza2, Christina Hartigan2, Melanie Donahue2, Katerina D. Popova2, Sebastian Koochaki1, Sergey V. Venev3, Jeanne Rivera5, EDwin Chen5, Kasper Lage2, Monica Schenone2, Alan D. D'AnDrea4, Steven A. Carr2, Elizabeth A. Morgan6, Job Dekker3,7, Benjamin L. Ebert1,2,7* 1 SUPPLEMENTAL METHODS Generation of cohesin-mutant single cell clones Both U937 anD K562 parental cell lines were confirmeD to be wilD type for all cohesin complex components and regulators based on RNA-Seq, anD exome or genome sequencing Data generated by the Cancer Cell Line Encyclopedia (CCLE) prior to development of any cohesin- mutant moDels(1). Of note, since STAG2 is an X-linkeD gene which unDergoes normal X- inactivation in females, patients with preDicted loss-of-function (LOF) STAG2 mutations are predicted to lack normal STAG2 expression. Therefore, in both cases of U937 cells and K562 cells, which both carry two copies of the X chromosome, we screened for presence of homozygous STAG2 LOF mutations. U937- and K562-Cas9 expressing cells were first generateD by lentiviral transduction of parental U937 and K562 cells with a lentiviral vector expressing the Cas9 nuclease unDer blasticiDin selection (pLX-311Cas9; Addgene #96924). Each Cas9- expressing cell line was subjected to a Cas9 activity assay using a separate lentiviral transduction with a GFP reporter construct (pXPR-011; Addgene #59702) and only cell lines with >80% Cas9 activity as determined by flow cytometry were used for subsequent generation of single cell clones. To generate the single cell clones, we cloned sgRNAs targeting STAG2, SMC3, RAD21 or non-targeting sgRNAs into a minimal backbone plasmiD (ADDgene #41824) and transfected into U937-Cas9 or K562-Cas9 cells using nucleofection (Lonza, Nucleofector II). We single cell sorted GFP+ cells into 96 well plates 36-48 hours after transfection, and grew up single cell clones, which we confirmed by DNA sequencing of the targeted locus, as well as Western blotting. Select cohesin-mutant clones were transDuceD with GFP or mCherry anD luciferase containing lentiviral plasmids (lenti-GFP-Luc2, or lenti-mCherry-Luc2) and sorted for GFP or mCherry positivity for use in mouse transplant experiments and competition assays. Choice of sgRNAs was Driven by observation of presumeD pathogenic STAG2 mutations in patients baseD on RapiD Heme Panel sequencing at the Dana-Farber Cancer Institute (DFCI cBioPortal) (2). Western Blotting and Immunoprecipitation (IP) Cells were washeD in colD 1X PBS (Corning CellGro) anD lyseD for 15-30min on ice using NP-40 lysis buffer (150mM NaCl, 50mM Tris pH 7.5 (Thermo Fisher Scientific 15567027), 1% NP40 (Roche 11332473001), 5% glycerol (Sigma-AlDrich G5516), anD 100X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific 78447) in water). Lysates were quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 23225) and reduced with 6X Laemmli SDS-Sample Buffer (Boston BioProDucts BP-111R) and 10X NuPAGE Sample ReDucing Agent (Thermo Fisher Scientific NP0009) for 5min at 95°C. For SMC1A IPs, protein lysates were incubateD with SMC1A antiboDy overnight rotating at 4°C. 50µL of Protein G or A agarose beads in a 50% slurry (EZview Red Protein G Affinity Gel, Sigma- AlDrich, E3403 anD EZview ReD Protein A Affinity Gel, Sigma-AlDrich, P6486) were washeD three times with NP-40 lysis buffer and incubated with the antibody and lysate mixture. The mixture was rotateD for 2hrs at 4°C, anD the beaDs were then washed once with NP-40 lysis buffer and twice with wash buffer (150mM NaCl, 50mM Tris pH 7.5 (Thermo Fisher Scientific 15567027), 1% glycerol (Sigma-AlDrich G5516), 100X Halt Protease anD Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific 78447) in water). Samples were reDuceD as stateD above. For Western blotting, samples were run on NuPAGE 4-12% Bis-Tris Protein Gels (Invitrogen NP0321 anD NP0316) in NuPAGE MOPS SDS Running Buffer 20X (Life Technologies NP0001) or NuPAGE MES SDS Running Buffer 20X (Life Technologies NP0002) and transferred onto Nitrocellulose Pre-Cut Blotting Membranes (0.45µm, Invitrogen LC2001) using 10X Tris/Glycine 2 Buffer (Bio-RaD 1610734) anD 20% Methanol (VWR TXBJ349664LBRI) in water. Gels were transferreD at 90V for 2-2.5hrs or 40V overnight at 4°C. Membranes were blocked with 5% non- fat Dry milk (Bio-RaD 1706404XTU) in Tris BuffereD Saline with Tween-20 (Cell Signaling Technology, 9997) for 30-60min at room temperature. Incubation with primary antibody was performed overnight at 4°C or for 2-4hrs at room temperature in 5% milk in TBST. Primary antibodies used for characterization of control and STAG2 knockout clones are as follows: Phospho-Histone H2A.X, STAG2, anti-SA1, anD anti-SMC1. Membranes were washeD 3 times for 5min each in TBST and incubated in Anti-rabbit IgG, Anti-mouse IgG, or Rabbit anti-Goat IgG secondary as needed in 5% milk in TBST for 1hr at room temperature. Membranes were washed an additional 3 times for 5min each in TBST and visualized after a 5min incubation with SuperSignal West Dura ExtenDeD Duration Substrate (Thermo Scientific, 34075), SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34078), or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232). AdDitional blots were imageD using the LI-COR platform (LI-COR Biosciences) with IRDye 800CW anD IRDye680RD secondary antibodies (mouse, IRDye 680LT Donkey-anti-mouse IgG(925-68022) used at 1:15,000; rabbit, IRDye 800CW Goat anti-rabbit IgG (926-32211) used at 1:10,000) and imaging was performeD on an ODyssey CLx InfrareD Imaging System. To assess levels of DNA Damage, cells were treateD with 100nM Talazoparib (Selleck S7048) and incubated at 37°C for 24hrs before harvesting protein lysate. Cells were also irraDiateD using an X-ray irraDiator at 100raDs or 10Gy, anD protein lysate was harvesteD after 1hr at 37°C. Samples were run on NuPAGE 3-8% Tris-Acetate Protein Gels (Thermo Fisher Scientific, EA0375) anD transferreD as above at 90V for 2hrs at 4°C. Membranes were blockeD for 30min at room temperature anD incubateD with the primary antiboDy overnight at 4°C. Primary antiboDies used are as follows: ATR, Chk1, Phospho-Chk2, anD Vinculin in 5% milk in TBST; Anti-Chk2 in 1% milk in TBST; Phospho-ATR, ATM , Phospho-ATM, anD Phospho-Chk1 in 5% BSA in TBST. Membranes were washeD 10 times for 10min each anD incubateD with the seconDary antiboDy as stated above for 1hr at room temperature. After incubation, membranes were washed an additional 10 times for 10min each and visualized as above. The following antiboDies were useD in the stuDy: Anti-ATR (Abcam, Cat#ab54793) Anti-SA1 (Abcam, Cat#ab4455) Anti-SMC1 (Abcam, Cat#ab140493) STAG2 (Cell Signaling Technology, Cat#4239) β-Actin, 8H10D10 (Cell Signaling Technology, Cat#3700) Phospho-Histone H2A.X, Ser139, 20E3 (Cell Signaling Technology, Cat#9718) Chk1, 2G1D5 (Cell Signaling Technology, Cat#2360) Phospho-Chk2, Th468, C13C1 (Cell Signaling Technology, Cat#2197) Phospho-ATR, Ser428, D6H9 (Cell Signaling Technology, Cat#2853) ATM, 11G12 (Cell Signaling Technology, Cat#92356) Phospho-ATM, Ser1981, D6H9 (Cell Signaling Technology, Cat#5883) Phospho-Chk1, ser345, 133D3 (Cell Signaling Technology, Cat#2348) Vinculin, E1E9V XP (Cell Signaling Technology, Cat#13901) Anti-rabbit IgG, HRP-linkeD (Cell Signaling Technology, Cat#7074) Anti-mouse IgG, HRP-linkeD (Cell Signaling Technology, Cat#7076) Anti-Chk2, clone 7 (EMD Millipore, Cat#05-649) Rabbit anti-Goat IgG, HRP conjugate (EMD Millipore, Cat#AP106P) 3 Genome-wide CRISPR screening 5 STAG2-mutant anD 6 STAG2-wilD type U937 cells expressing Cas9 anD confirmeD to have >80% Cas9 activity by the GFP reporter construct (pXPR-011; Addgene #59702) were infecteD with the genome-wiDe human Avana LentiGuiDe-Puro CRISPR library (BroaD Genetic Perturbations Platform), which contains ~75,000 sgRNAs targeting ~19,000 genes anD 1000 controls, in two separate experiments, as previously described (3). Briefly, cells were selecteD in blasticidin for 14 Days to maximize Cas9 expression. BlasticiDin was washeD out of the meDia two days prior to transduction. Puromycin selection of transduced cells was initiated on Day 2 after transDuction anD carrieD out for a total of 5 Days. BlasticiDin was restarteD on Day 8 after transDuction anD incluDeD in the meDia through the enD of the experiment. Cell pellets were collected on Day 21, genomic DNA was purified and the sgRNA sequences amplified with sufficient gDNA to maintain representation, and quantified using next generation sequencing. Transplantation assays and mouse analysis. 8-10 week old male and female NSGS mice (NOD-SCID; IL2Rγ null; Tg(IL3, CSF2, KITL)) obtained from The Jackson laboratory, strain 013062) were sublethally irradiated using Gamma- cell irradiator (Best Theratronics) at a dose of 250 Rads, and retro-orbitally injected with 200,000- 1 million cells/mouse. 4-10 recipients were injected per arm. Animals were monitored daily for presence of disease, and were sacrificed at designated times after transplantation or when moribunD. Peripheral blooD was collecteD from the retro-orbital cavity using a heparinized glass capillary and automated total and differential blood cell counts were determined using ADVIA Hematology system (Bayer). Collected blood was also used to prepare blood smears, which were stained with May Gruenwald and Modified Giemsa (Sigma Aldrich). Following sacrifice, mice were examined for presence of tumors, enlarged lymph nodes or other abnormalities, and organs were collecteD for further cell anD histopathologic analysis. Single cell suspensions were maDe from bone marrow, and spleen, washed, red blood cells were lysed, and samples were frozen in 10% dimethylsulfoxide/ 90% FCS until analysis.
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